Effect of free IR820 and IR820-PLGA NPs on metabolic activity

To evaluate the toxicity of free IR820 and IR820-PLGA NPs in the dark using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cells were seeded in a 96-well plate at 1 × 10⁴ cells per well and incubated overnight. Cells were treated with 0, 5, 10, 15, 25, or 35 μM of free IR820 dye or IR820-PLGA NPs for 4 h. Then, the cells were washed with PBS and incubated in fresh media for an additional 24 h prior to being incubated in MTT solution per the manufacturer’s instructions (Thermo Fisher). After 3 h, the MTT solution was replaced with dimethyl sulfoxide (DMSO) and the absorbance at 540 nm was read on a Synergy H1 plate reader (BioTek). To analyze the data, background (DMSO in wells without cells) was subtracted from the absorbance reading in each well. Triplicate well signals were averaged and then normalized to untreated cells. These experiments were performed in triplicate and analyzed by Student’s t tests at each concentration.